GSM7749112: A375 Day14 rep2; Homo sapiens; OTHER
To extract gDNA, screen cells were lysed as in (Sidi Chen et al. 2015) with 12 ml of NK lysis buffer for 100 million cells (50 mM Tris, 50 mM ethylenediaminetetraacetic acid, 1% SDS and pH 8). Once cells were resuspended, 60 µl of 20 mg/ml Proteinase K (Qiagen) was added and the sample was incubated at 55 °C overnight. The next day, 60 µl of 20 mg/ml RNase A (Qiagen) was added and mixed, and samples were incubated at 37 °C for 30 min. Then, 4 ml of prechilled 7.5 M ammonium acetate was added, and samples were vortexed and spun at 4,000g for 10 min. The supernatant was placed in a new tube, mixed well with 12 ml isopropanol and spun at 4,000g for 10 min. DNA pellets were washed with 12 ml of 70% ethanol, spun and dried, and pellets were resuspended with 0.2× TE buffer (Sigma-Aldrich). In addition, we also generated linearized plasmid library input and diluted it down to mimic similar copy number conditions as the gDNA samples. To amplify the gRNA cassette from gDNA and add indexing for Illumina sequencing, we used a two-step PCR protocol, PCR1 and PCR2, respectively. For the PCR1 reaction, we used 960 µg gDNA for each sample. We performed 96× 100 µl PCR1 reactions per sample (10 µl 10× Taq buffer, 0.02 U/µl Taq-B enzyme (Enzymatics, P7250L), 0.2 mM dNTPs, 0.2 µM forward and reverse primers and 100 ng gDNA per µl). Thermocycler conditions were 94 °C/30 s, 20× (94 °C/10 s, 55 °C/30 s, 68 °C/45 s) and 68 °C/3 min. For each sample, all PCR1 products were pooled and mixed. For the PCR2 reaction, we performed 2 reactions per sample (20 µl 5× NEB Q5 buffer, 0.01 U/µl Q5 enzyme, 20 µl PCR1 product, 0.2 mM dNTPs and 0.4 µM forward and reverse PCR2 primers in 100 µl). Thermocycling conditions for PCR2 were 98 °C/30 s, 7× (98 °C/10 s, 63 °C/30 s and 72 °C/45 s) and 72 °C/5 min. For each sample, PCR2 products were pooled, followed by normalization (gel-based band densitometry quantification), before combining equal amounts of uniquely barcoded samples.    The pooled product was then purified using SPRI beads. First, we performed a 0.6× vol/vol SPRI to remove gDNA carryover, followed by the addition of a 0.3× vol/vol SPRI (0.6 + 0.3 = 0.9× final) to the supernatant to purify the ~260 bp PCR product. The final amplicons were sequenced on Illumina NextSeq 500-II HighOutput 1 × 150 v2.5 (Cat #20024907).